The environmental pollution by endocrine disruptors becomes problems, and research of the state of pollution, investigation of the influence on the health of human beings and the like have been carried out. As the influences of these endocrine disruptors on human beings and on the environment are revealed, they became great social concerns not only in Japan but also in many countries of the world. Among others, dioxins are suspected of lasting influences on human beings and on the ecosystem and environment, and therefore, research of the state of pollution, investigation of the state of exposure in human beings and in the ecosystem and elucidation of intake routes as well as development of a method of monitoring the amount of dioxins in polluted locations and a method of removing the pollution are accelerated. Since dioxins are formed, for example, in the course of use, production and combustion of organic chlorine compounds, the sources of the dioxins are wide-ranging and a widespread pollution is confirmed in soil, water, atmosphere, foods, marine products and the like. Accordingly, it is desired to establish a simple and rapid method for determining dioxins in samples, owing to the need of determining dioxin concentrations in numerous samples such as biological and environmental samples, and of taking measures to the pollution.
Dioxins include a number of congeners comprised of 75 kinds of polychlorodibenzodioxins (PCDDs) and 135 kinds of polychlorodibenzofurans (PCDFs). The relative toxicity of each dioxin congener, when assuming that the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) having the highest toxicity is 1, is shown as Toxic Equivalency Factor, and 7 kinds of PCDDs and 10 kinds of PCDFs having a high toxicity are used as object substances for measurement in the analysis of dioxins. Also, of polychlorobiphenyls (PCBs) which are recognized as one group of endocrine disruptors and are seen as a problem for some time, 12 kinds of co-planar PCBs have been measured as dioxins.
Previously, the determination of dioxins has been carried out by means of a high-resolution gas chromatography/mass spectrometry (HRGC/HRMS) analysis. However, the HRGC/HRMS method needs a multi-stage of complicated cleanup procedures to remove interfering substances contained in samples, an expensive analytical instrument, and skilled analyzers. Accordingly, the use of the method is limited to an analysis in a particular analytical facility. In analytical methods of dioxins, in particular, in the RGC/HRMS method, the contents of 17 kinds of dioxin congeners each having a high toxicity value are individually quantified, and the actually quantified content of each congener is then multiplied by the corresponding Toxic Equivalency Factor. Then, the sum total of the values obtained for all the congeners is calculated as Toxic Equivalent Quantity (TEQ) which corresponds to the amount of 2,3,7,8-TCDD, and the TEQ values are used as analytical values of the dioxins. Thus, the method needs much time for analysis of samples involving data analysis. For the above reasons, it is strongly desired to develop a convenient, cheep and highly sensitive method for determining dioxins in samples.
On the other hand, there persistently exists an idea that the dioxin amount (TEQ) is more simply determined by measuring a particular indicator substance. One of these methods is that measuring chlorobenzene which is a precursor of dioxins. Recently, it has been proved that the amount of 2,3,4,7,8-PeCDF, which is one of dioxin congeners, has a very high correlation with the total TEQ value of dioxins (Takasuga et al., 11th Symposium on Environmental Chemistry, Program and Abstracts, p. 136, 2002). In a wide range of samples, for example, environmental samples such as soil, mud, atmosphere, water, exhaust gas and ash, biological samples such as mother's milk and blood as well as marine products, foods and the like, 2,3,4,7,8-PeCDF is a major constituent in all dioxins, and its content shows a high correlation (R=0.96-0.99) with the total TEQ value of dioxins. Accordingly, attention is paid to 2,3,4,7,8-PeCDF as an indicator substance for investigating the amount of dioxins.
On the other hand, an attempt to quantify the dioxins using an antibody is also made in the art.
For example, JP-A-2002/340882 discloses a method for determining dioxins and an apparatus therefor comprising four units, i.e. a collection unit, an extraction unit, a separation and purification unit, and a measurement unit for determining dioxins using an antibody.
Also, JP-A-2002/228660 discloses a method for detecting dioxins contained in biological samples such as human blood and mother's milk by preparing and using a monoclonal antibody having a high affinity for 2,3,7,8-TCDD.
In addition, JP-A-2002/119279 discloses a method for deducing an amount of dioxins present in samples using a few antibodies having a cross-reactivity with plural congeners within the range of dioxin.
However, these references do not disclose monoclonal antibodies recognizing 2,3,4,7,8-PeCDF, as well as, gene sequences encoding said monoclonal antibodies, recombinant antibodies based on said gene sequences and a method for determining 2,3,4,7,8-PeCDF using said recombinant antibodies.
Also, methods disclosed in these references have a disadvantage that they are insufficient for investigating the TEQ values of dioxins contained in samples.